Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.     

Identification of nicotinic acetylcholine receptor amino acids photolabeled by the volatile anesthetic halothane

To identify inhalational anesthetic binding domains in a ligand-gated ion channel, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with [(14)C]halothane and determined by Edman degradation some of the photolabeled amino acids in nAChR subunit fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography. Irradiation at 254 nm for 60 s in the presence of 1 mM [(14)C]halothane resulted in incorporation of approximately 0.5 mol of (14)C/mol of subunit, with photolabeling distributed within the nAChR extracellular and transmembrane domains, primarily at tyrosines. GammaTyr-111 in ACh binding site segment E was labeled, while alphaTyr-93 in segment A was not. Within the transmembrane domain, alphaTyr-213 within alphaM1 and deltaTyr-228 within deltaM1 were photolabeled, while no labeled amino acids were identified within the deltaM2 ion channel domain. Although the efficiency of photolabeling at the subunit level was unaffected by agonist, competitive antagonist, or isoflurane, state-dependent photolabeling was seen in a delta subunit fragment beginning at deltaPhe-206. Labeling of deltaTyr-212 in the extracellular domain was inhibited >90% by d-tubocurarine, whereas addition of either carbamylcholine or isoflurane had no effect. Within M1, the level of photolabeling of deltaTyr-228 with [(14)C]halothane was increased by carbamylcholine (90%) or d-tubocurarine (50%), but it was inhibited by isoflurane (40%). Within the structure of the nAChR transmembrane domain, deltaTyr-228 projects into an extracellular, water accessible pocket formed by amino acids from the deltaM1-deltaM3 alpha-helices. Halothane photolabeling of deltaTyr-228 provides initial evidence that halothane and isoflurane bind within this pocket with occupancy or access increased in the nAChR desensitized state compared to the closed channel state. Halothane binding at this site may contribute to the functional inhibition of nAChRs.     

Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic

To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [(3)H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [(3)H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC(50) = 70 microm) than in the closed channel state. In addition, both drugs between 0.1 and 1 mm produced a concentration-dependent enhancement of [(3)H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential [(3)H]azietomidate photoincorporation into the nAChR alpha and delta subunits. Photolabeled amino acids in both subunits were identified in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized state, there was labeling of alphaGlu-262 and deltaGln-276 at the extracellular end and deltaSer-258 and deltaSer-262 toward the cytoplasmic end. Within the acetylcholine binding sites, [(3)H]azietomidate photolabeled alphaTyr-93, alphaTyr-190, and alphaTyr-198 in the site at the alpha-gamma interface and deltaAsp-59 (but not the homologous position, gammaGlu-57). Increasing [(3)H]azietomidate concentration from 1.8 to 150 microm increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [(3)H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.     

Bupropion binds to two sites in the Torpedo nicotinic acetylcholine receptor transmembrane domain: a photoaffinity labeling study with the bupropion analogue [(125)I]-SADU-3-72

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone (SADU-3-72). Based on inhibition of [(125)I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC(50) = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC(50) = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [(125)I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [(125)I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu(265), βLeu(257)) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr(213) in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.     

Identification of the sites of incorporation of [3H]ethidium diazide within the Torpedo nicotinic acetylcholine receptor ion channel

The binding sites of ethidium, a noncompetitive antagonist of the nicotinic acetylcholine receptor (nAChR), have been localized in the Torpedo nAChR in the desensitized state by use of a photoactivatible derivative, [(3)H]ethidium diazide. At 10 microM [(3)H]ethidium diazide, incorporation into the alpha-, beta-, and delta-subunits was inhibited by the presence of phencyclidine (PCP). Within the alpha-subunit, the incorporation was mapped to a 20-kDa fragment beginning at alphaSer-173 and containing the first three transmembrane segments, alphaM1, alphaM2, and alphaM3. Further digestion of this fragment generated two fragments with PCP-inhibitable incorporation, one containing alphaM1 and one containing both alphaM2 and alphaM3. Within alphaM2, specific incorporation was present in alphaLeu-251 and alphaSer-252, residues that have been previously shown to line the lumen of the ion channel. Digestion of the delta-subunit with S. aureus V8 protease generated a 14-kDa and a 20-kDa fragment, both of which began at Ile-192 and contained PCP-inhibitable labeling. The 14-kDa fragment, containing deltaM1 and deltaM2, was further digested to generate a 3-kDa fragment, containing deltaM2 alone, with PCP-inhibitable incorporation. Digestion of the 20-kDa fragment, which contained deltaM1, deltaM2, and deltaM3, generated two fragments with incorporation, one containing the deltaM1 segment and the other containing deltaM2 and deltaM3. These results establish that in the desensitized state of the nAChR, the high-affinity binding site of ethidium is within the lumen of the ion channel and that the bound drug is in contact with amino acids from both the M1 and M2 hydrophobic segments.     

Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.     

Identification of sites of incorporation in the nicotinic acetylcholine receptor of a photoactivatible general anesthetic

Most general anesthetics including long chain aliphatic alcohols act as noncompetitive antagonists of the nicotinic acetylcholine receptor (nAChR). To locate the sites of interaction of a long chain alcohol with the Torpedo nAChR, we have used the photoactivatible alcohol 3-[(3)H]azioctanol, which inhibits the nAChR and photoincorporates into nAChR subunits. At 1 and 275 microm, 3-[(3)H]azioctanol photoincorporated into nAChR subunits with increased incorporation in the alpha-subunit in the desensitized state. The incorporation into the alpha-subunit was mapped to two large proteolytic fragments. One fragment of approximately 20 kDa (alpha V8-20), containing the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of approximately 10 kDa (alpha V8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within alpha V8-20, the primary site of incorporation was alpha Glu-262 at the C-terminal end of alpha M2, labeled preferentially in the desensitized state. The incorporation at alpha Glu-262 approached saturation between 1 microm, with approximately 6% labeled, and 275 microm, with approximately 30% labeled. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface at approximately 1% of the levels in alpha Glu-262. Therefore, the primary binding site of 3-azioctanol is within the ion channel with additional lower affinity interactions within the agonist binding site and at the protein-lipid interface.     

Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [³H]AziPm, and labeled amino acids were identified by Edman degradation. [³H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18′ and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (−agonist) amino acids in the M2 helices (αM2-6′, βM2-6′ and -13′, and δM2-13′) that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10′. Propofol enhanced [³H]AziPm photolabeling at αM2-10′. Propofol inhibited [³H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC₅₀ = 40 μm) than it inhibited ion channel photolabeling (IC₅₀ = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.     

Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor

Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate).     

Photolabeling the Torpedo nicotinic acetylcholine receptor with 4-azido-2,3,5,6-tetrafluorobenzoylcholine, a partial agonist

The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (K(eq) = 12 microM) for the two agonist binding sites. Upon UV irradiation at 254 nm, [3H]APFBzcholine was photoincorporated into the nAChR alpha, gamma, and delta subunits in an agonist-inhibitable manner. Photolabeled amino acids in the agonist binding sites were identified by Edman degradation of isolated, labeled subunit fragments. [3H]APFBzcholine photolabeled gammaLeu-109/deltaLeu-111, gammaTyr-111, and gammaTyr-117 in binding site segment E as well as alphaTyr-198 in alpha subunit binding site segment C. The observed pattern of photolabeling is examined in relation to the predicted orientation of the azide when APFBzcholine is docked in the agonist binding site of a homology model of the nAChR extracellular domain based upon the structure of the snail acetylcholine binding protein.     

Identification of binding sites in the nicotinic acetylcholine receptor for TDBzl-etomidate, a photoreactive positive allosteric effector

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.     

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine,a novel photoaffinity antagonist

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.     

The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits

A binding site for the channel-blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP+) was localized in the alpha-, beta- and delta-chains of the nicotinic acetylcholine receptor (AChR) from Torpedo marmorata electric tissue. The photolabel was found in homologous positions of the highly conserved sequence helix II, alpha 248, beta 254, and delta 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position delta 262 independent of whether photolabeling was performed with the receptor in its resting, desensitized or antagonist state. A model of the AChR ion channel is proposed, according to which the channel is formed by the five helices II contributed by the five receptor subunits.     

Characterization of the transient agonist-triggered state of the acetylcholine receptor rapidly labeled by the noncompetitive blocker [3H]chlorpromazine: additional evidence for the open channel conformation

The kinetics of covalent labeling of the alpha, beta, gamma, and delta chains of the acetylcholine receptor (AcChR) from Torpedo marmorata by the noncompetitive blocker [3H]chlorpromazine ([3H]CPZ) are investigated by using rapid mixing photolabeling techniques. In an initial study [Heidmann, T., & Changeux, J. P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1897-1901], it was shown that the rate of [3H]CPZ labeling increases 100-1000-fold upon simultaneous addition of nicotinic agonists to the AcChR and that prior addition of these agonists abolishes the effect. The data were interpreted in terms of the rapid labeling of the transient active state of the AcChR where the ion channel is in its open configuration. This interpretation was recently challenged [Cox, R. N., Kaldany, R. R. J., Di Paola, M., & Karlin, A. (1985) J. Biol. Chem. 260, 7186-7193] on the ground of studies with a different noncompetitive blocker, [3H]quinacrine azide, and the suggestion was made that this compound labels the rapidly desensitized closed channel conformation of the AcChR. In this paper it is shown that the rate of rapid labeling of the AcChR by [3H]CPZ decreases to negligible values upon exposure of the AcChR to nicotinic agonists, in the 100-500-ms time range. The absolute values of the rate constants of this decrease (10-15 s-1 for saturating concentrations of acetylcholine and carbamoylcholine) and their variation with agonist concentration (apparent dissociation constants of 40 microM and 0.4 mM for acetylcholine and carbamoylcholine, respectively) are those expected for the rapid desensitization of the AcChR.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hydrophobic photoreagent 3-(trifluoromethyl)-3-m-([125I] iodophenyl) diazirine is a novel noncompetitive antagonist of the nicotinic acetylcholine receptor

We have shown previously that the lipophilic photoreagent 3-(trifluoromethyl)3-m-([125I]iodophenyl)-diazirine ([125I]TID) photolabels all four subunits of the Torpedo nicotinic acetylcholine receptor (AChR) and that greater than 70% of this photoincorporation is inhibited by cholinergic agonists and some noncompetitive antagonists, including histrionicotoxin (HTX), but not phencyclidine (PCP; White, B.H., and Cohen, J.B. (1988) Biochemistry 27, 8741-8751). We have now examined the effects of nonradioactive TID on (a) AChR photoincorporation of [125I]TID, (b) AChR-mediated ion transport, and (c) AChR binding of several cholinergic ligands. We find that TID inhibits [125I]TID photoincorporation into the AChR to the same extent as carbamylcholine. The saturable component of [125I]TID photolabeling is half-maximal at 4 microM [125I]TID with 0.5 mol specifically incorporated per mol of AChR after 30 min photolysis with 60 microM [125I]TID. Repeated labeling of membranes at a fixed [125I]TID concentration gave results consistent with a maximal incorporation of one [125I]TID molecule per AChR. Nonradioactive TID also noncompetitively inhibits agonist-stimulated 22Na+ efflux from Torpedo vesicles with an IC50 of 1 microM. Furthermore, TID inhibits allosterically the binding of [3H]HTX, decreasing its affinity for the AChR 5-fold both in the presence and absence of agonist. In contrast, TID has little effect on [3H]PCP binding in the absence of agonist but completely inhibits it in the presence of agonist. TID enhances the cooperativity of [3H]nicotine binding. [125I]TID is thus a photoaffinity label for a novel noncompetitive antagonist binding site on the AChR that is linked allosterically to the binding sites of both agonists and other noncompetitive antagonists. The [125I]TID site is presumably located within the central pore of the AChR.     

Examining the noncompetitive antagonist-binding site in the ion channel of the nicotinic acetylcholine receptor in the resting state

3-Trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) has been shown to be a potent noncompetitive antagonist (NCA) of the nicotinic acetylcholine receptor (AChR). Amino acids that contribute to the binding site for [(125)I]TID in the ion channel have been identified in both the resting and desensitized state of the AChR (White, B.H., and Cohen, J.B. (1992) J. Biol. Chem. 267, 15770-15783). To characterize further the structure of the NCA-binding site in the resting state channel, we have employed structural analogs of TID. The TID analogs were assessed by the following: 1) their ability to inhibit [(125)I]TID photoincorporation into the resting state channel; 2) the pattern, agonist sensitivity, and NCA inhibition of [(125)I]TID analog photoincorporation into AChR subunits. The addition of a primary alcohol group to TID has no demonstrable effect on the interaction of the compound with the resting state channel. However, conversion of the alcohol function to acetate, isobutyl acetate (TIDBIBA), or to trimethyl acetate leads to rightward shifts in the concentration-response curves for inhibition of [(125)I]TID photoincorporation into the AChR channel and a progressive reduction in the agonist sensitivity of [(125)I]TID analog photoincorporation into AChR subunits. Inhibition of [(125)I]TID analog photoincorporation by NCAs (e.g. tetracaine) as well as identification of the sites of [(125)I]TIDBIBA photoincorporation in the deltaM2 segment indicate a common binding locus for each TID analog. We conclude that relatively small additions to TID progressively reduce its ability to interact with the NCA site in the resting state channel. A model of the NCA site and resting state channel is presented.     

Cholesterol interacts with transmembrane alpha-helices M1, M3, and M4 of the Torpedo nicotinic acetylcholine receptor: photolabeling studies using [3H]Azicholesterol

The photoactivatable sterol probe [3alpha-(3)H]6-Azi-5alpha-cholestan-3beta-ol ([3H]Azicholesterol) was used to identify domains in the Torpedo californica nicotinic acetylcholine receptor (nAChR) that interact with cholesterol. [3H]Azicholesterol partitioned into nAChR-enriched membranes very efficiently (>98%), photoincorporated into nAChR subunits on an equal molar basis, and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine, consistent with photoincorporation at the nAChR lipid-protein interface. Sites of [3H]Azicholesterol incorporation in each nAChR subunit were initially mapped by Staphylococcus aureus V8 protease digestion to two relatively large homologous fragments that contain either the transmembrane segments M1-M2-M3 (e.g., alphaV8-20) or M4 (e.g., alphaV8-10). The distribution of [3H]Azicholesterol labeling between these two fragments (e.g., alphaV8-20, 29%; alphaV8-10, 71%), suggests that the M4 segment has the greatest interaction with membrane cholesterol. Photolabeled amino acid residues in each M4 segment were identified by Edman degradation of isolated tryptic fragments and generally correspond to acidic residues located at either end of each transmembrane helix (e.g., alphaAsp-407). [3H]Azicholesterol labeling was also mapped to peptides that contain either the M3 or M1 segment of each nAChR subunit. These results establish that cholesterol likely interacts with the M4, M3, and M1 segments of each subunit, and therefore, the cholesterol binding domain fully overlaps the lipid-protein interface of the nAChR.     

Structure of the agonist-binding sites of the Torpedo nicotinic acetylcholine receptor: affinity-labeling and mutational analyses identify gamma Tyr-111/delta Arg-113 as antagonist affinity determinants.

Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the gamma-subunit which, along with the analogous residue in delta-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and alpha-conotoxin (alpha Ctx) MI. nAChR gamma-subunit, isolated from nAChR-rich membranes photolabeled with [3H]dTC, was digested with Staphylococcus aureus V8 protease, and a 3H-labeled fragment was purified by reversed-phase high-performance liquid chromatography. Amino-terminal sequence analysis of this fragment identified 3H incorporation in gamma Tyr-111 and gamma Tyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at gamma Trp-55, the primary site of [3H]dTC photoincorporation within gamma-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940-32950]. The Torpedo nAChR delta-subunit residue corresponding to gamma Tyr-111 (delta Arg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the alpha-delta agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]alpha-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and alpha Ctx MI with nAChRs containing gamma Y111R or delta R113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the gamma Y111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of alpha Ctx MI, with delta R113Y enhancing and gamma Y111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of gamma- (or delta-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.     

Decidium. A novel fluorescent probe of the agonist/antagonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor

We have examined the interaction of the nicotinic acetylcholine receptor with decidium diiodide, a bisquaternary analogue of ethidium containing 10 methylene groups between the endocyclic and trimethylamino quaternary nitrogens. Decidium inhibits mono-[125I]iodo-alpha-toxin binding, inhibits agonist-elicited 22Na+ influx in intact cells, augments agonist competition with mono-[125I]iodo-alpha-toxin binding, and enhances [3H]phencyclidine (PCP) binding to a noncompetitive inhibitor site. These effects occur over similar concentration ranges (half-maximum effects between 0.1 and 0.4 microM). Thus, decidium binds to the agonist site and converts the receptor to a desensitized state exhibiting increased affinity for agonist and heterotropic inhibitors. These properties are similar to metaphilic antagonists characterized in classical pharmacology. At higher concentrations decidium associates directly with the noncompetitive inhibitor site identified by [3H]phencyclidine binding. Dissociation constants of decidium at this site in the resting and desensitized states are determined to be 29 and 1.2 microM, respectively. Analysis of fluorescence excitation and emission maxima reveal that binding to both the agonist and noncompetitive inhibitor sites is associated with approximately 2-fold enhancement of fluorescence. The excitation maximum for decidium bound at the agonist site appears at 490 nm while that for decidium bound at the noncompetitive inhibitor site appears at 530 compared to 480 nm in buffer. These results suggest that decidium experiences a more hydrophobic environment upon binding to the nicotinic acetylcholine receptor sites, particularly to the noncompetitive inhibitor site. Fluorescence energy transfer between N'-fluorescein isothiocyanate-lysine-23 alpha-toxin (FITC-toxin), and decidium is not detected when each is bound to one of the two agonist sites on the receptor. This allows a minimal distance to be estimated between fluorophores. In contrast, energy transfer is observed between decidium nonspecifically associated with the membrane or with nonspecific sites and the FITC-toxin at the agonist sites.     

Mapping the structural requirements for nicotinic acetylcholine receptor activation by using tethered alkyltrimethylammonium agonists and antagonists

A molecule as simple in structure as tetramethylammonium gates the nicotinic acetylcholine receptor (nAChR) with high efficacy. To compare the structure of the nAChR transmitter binding site in the open channel state with that of the ACh binding protein, we determined the efficacy of nAChR gating by -S(CH(2))(n)N(CH(3))(3)(+) (n = 1-4) tethered to substituted cysteines at positions in the alpha subunits or gamma and delta subunits predicted to contribute to the ACh binding sites in mutant Torpedo nAChRs expressed in Xenopus oocytes. For tethered thiocholine [-S(CH(2))(2)N(CH(3))(3)(+)], we previously reported that within alpha195-201 gating was observed only at alphaY198C while at alphaY93C it acted as an antagonist. We now show that within alpha191-194, thiocholine activates when tethered at alphaCys192 or alphaCys193. Thiocholine also activates when tethered at alphaY190C or alphaW149C in nAChRs containing a beta subunit mutation (betaL257S) that destabilizes the closed channel, but not from gammaW55C/deltaW57C, where longer adducts can activate. When tethered at positions in binding site segment E, thiocholine activates only from gammaL119C/deltaL121C, where the shorter -S(CH(2))(1)N(CH3)(3)(+) acts as an antagonist. Longer adducts tethered at gammaL109C/deltaL111C or gammaL119C/deltaL121C also activate, but less efficiently. The length requirements for efficient gating by tethered agonists agree closely with predictions based upon the structure of the agonist site in a nAChR homology model derived from the ACh binding protein structure, which suggests that this structure is an excellent model of the nAChR agonist binding site in the open channel conformation. The inability of thiocholine to activate from alphaY93C, which is not predicted by the model, is discussed in terms of the structure of the nAChR in the closed state.